UNIT-1Gene Cloning

Presented By:-Ms. Smita Shukla

Assistant ProfessorN.I.E.T, Greater Noida

What is a Gene ?

• Gene is a hereditary unit consisting of a sequence of DNA that occupies a specific location on a chromosome and determines a particular characteristic in an organism.

WHAT IS CLONING ?• Cloning in Biotechnology refers to processes

used to create identical copies of DNA fragments , cells or organisms .

* Clone is a group of cells derived from a single

parental cell by asexual reproduction

These identical copies are called clones

Cloning refers to DNA fragments - molecular or gene cloning

Cloning refers to cells – cell cloning

Cloning refers to organism - organism cloning

What is gene cloning ?• To "clone a gene" is to make

many copies of it

• Act of making many identical copies of gene

• Gene can be an exact copy of a natural gene

• Gene can be an altered version of a natural gene

Whole organisms are cloned too, but differently

BASIC STEPS IN GENE CLONING

• Preparation of Pure sample of DNA from the organism of desire

• Cutting of the DNA molecule using Restriction enzymes to form fragments

• Analysis of DNA fragments using electrophoresis

The action of a restriction enzyme ,EcoRI

Steps in Cloning a Gene• The First step in cloning a gene is to isolate the

DNA from the organism that contains the desired gene.

Steps in Cloning a Gene• The isolated DNA is purified and then

fragmented with a restriction enzyme.

• Restriction enzymes used in cloning produce staggered cuts in specific sequences in the DNA, generating fragments with the cohesive ends.

Steps in Cloning a Gene

• Each fragment has a single stranded sequence of nucleotides on its ends that is capable of hybridizing with DNA that has been fragmented with the same restriction enzyme.

• The DNA fragments are then incorporated into the plasmids.

• The type of plasmid used for cloning has a single restriction site, and when cleaved by the restriction enzyme, generates the same cohesive ends that are in the fragments of DNA to be cloned.

• The cohesive ends of the plasmid and DNA fragments now line up and the enzyme, DNA ligase, is used to form Phosphodiester bonds.

• The next step is to incorporate the plasmid into bacterial host cells by transformation.

• Each cell contains a different segment of DNA from the original organism. Taken together these cells represent a DNA library.

• The cells can now be plated out on agar medium.

• The colony of cells containing the desired cloned gene can then be identified and isolated.

INFERENCE• Within the host cell the vector multiplies and

produces numerous identical copies not only of itself but also of the gene that it carries.

• After that the host cell divides … as the recombinant vector replicates

• Now colony or clone, of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule

• The gene is cloned.

Application of Bacteria & Virus used in Genetic

Engineering

What is genetic engineering?

• Genetic engineering refers to the direct manipulation of DNA to alter an organism’s characteristics in a particular way.

• This may mean changing one base pair (A-T or C-G), deleting a whole region of DNA, or introducing an additional copy of a gene.

• It may also mean extracting DNA from another organism’s genome and combining it with the DNA of that individual.

• A gene is much too small to be inserted with some kind of microsurgery.

• Therefore some carrier ("vector") is required which takes the gene into the recipient cell and somehow gets it inserted into the DNA of the recipient.

Methods for gene insertion• Different means are used for carrying the desired

gene into the hereditary substance..

• For plants, the most common method is the use of a bacterium, Agrobacterium tumefaciens.

• For animals, certain viruses are used.

1. Bacteria as gene carriers

PLASMIDS

A small circles of DNA found in bacteria andsome other organisms.• Plasmids can replicate independently of the

host cell .

Desirable properties of a Vector

• It should be small.• Its DNA sequence should be known.• It should grow to high copy number in the host

cell.• It should contain a selectable marker that allows

easy selection of transformed host cell.• It should replicate autonomously• Should be easy to isolate & purify• Should contain one unique restriction enzymes.

• Also, when expression of DNA is needed then expression vectors are used.

• Expression vectors should contain control elements; promoter, operator etc..

Types of vectors• Choice of vector is dependent on insert size and

application.

• For Bacterial Cells:• Plasmids• Bacteriophage( Lambda phage & M 13 Phage)• Cosmids• Phagemids• YAC( Yeast Cloning Vectors)

For Plant cells:Binary Vector systemCo -integrative vector system

For Animal/ Mammalian cells:RetrovirusSV40 Viral VectorEBV( Epstein- Barr Virus), BPV( Bovine Papilloma

Virus)

Cloning VectorContains: • An origin of replication

• Selectable marker gene ( eg. Ampicillin resistance gene) and

• Multiple cloning site containing unique restriction enzymes sites..

Plasmid• A small circular DNA molecule found in

bacteria.• Used as cloning vectors. • Example: PUC19

Plasmid classification• Fertility or F – plasmids

• Resistance or R plasmids

• Col plasmids

• Degradative plasmids

• Virulence

2. Bacteriophages

• Phages are very simple in structure,

• Consisting of a DNA molecule carrying a number of genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein molecules

• Examples: T2, T4 and T6 Bacteriophages that infect E.coli.

• Filamentous phages with single stranded DNA such as M13

• Transfer of genetic elements from one bacterium to another by a bacteriophage is termed as transduction.

Why is Bacteria used for genetic engineering?

• Generation time is very small that is 20 minutes.

• Bacteria's DNA is very simple and can easily be transplanted from one bacterium to another

Cloning Process

How are viruses used in Genetic Engineering

• Viruses are tiny, nonliving particles that must have a host cell to replicate and flourish.

• Ability to attach to and invade specific cells, and incorporate the DNA (and/or RNA) they are carrying into the host cell, where it combines with the host cell's DNA.